The conjugation of water-soluble polyalkylene oxides with therapeutic moieties such as proteins and polypeptides is known. See, for example, U.S. Pat. No. 4,179,337, the disclosure of which is hereby incorporated by reference. The '337 patent discloses that physiologically active polypeptides modified with PEG circulate for extended periods in vivo, have reduced immunogenicity and antigenicity.
To conjugate polyalkylene oxides, the hydroxyl end-groups of the polymer must first be converted into reactive functional groups. This process is frequently referred to as "activation" and the product is called an "activated polyalkylene oxide."
For the most part, research has been directed to covalent attachment of polyalkylene oxides (PAO's) to epsilon amino groups of proteins, enzymes and polypeptides. Covalent attachment of polyalkylene oxides to lysine amino groups has been effected by linking groups such as succinoyl-N-hydroxysuccinimide ester, as disclosed by Abuchowski et al., Cancer Biochem. Biophys., 7, 175-86 (1984), azlactones, aryl imidates and cyclic imide thiones. See U.S. Pat. Nos. 5,298,643, 5,321,095, and 5,349,001, for example. The contents of each of the foregoing patents are hereby incorporated by reference herein. PAO's have also been activated with hydrazine groups in order to couple the polymer to activated carbohydrate groups.
In addition to the foregoing, the conversion of terminal hydroxy groups of PAO's such as PEG to carboxylic acids has also been reported. PEG-acids are useful in at least two regards. First, carboxylic acid derivatives can be used directly to conjugate nucleophiles via available hydroxyl or amino moieties. Secondly, PAO carboxylic acids can be used as intermediates to form other types of activated polymers. For example, mPEG carboxylic acids can be converted to the succinimidyl ester derivative via N-hydroxysuccinimide and a condensing agent such as diisopropyl carbodiimide. Other activated PAO's can be prepared by reaction of the active ester with hydrazine to produce PAO-hydrazide derivatives.
The principal drawback in preparing carboxylic add derivative of polyalkylene oxides has been the difficulty in obtaining high yields of pure product. For example, Journal of Controlled Release, 10 (1989) 145-154 and Polymer Bulletin, 18, (1987), 487-493, describe the synthesis of mPEG acids by converting mPEG-OH to an ethyl ester followed by base catalyzed hydrolysis to form the carboxylic acid. Ostensibly, this classic approach should proceed without difficulty. In realty, however, this method at best provides m-PEG acids of about 90% purity, with the main product contaminant being the starting material, mPEG-OH. In addition, the separation of the desired PEG add from the starting PEG alcohol is very difficult. Standard laboratory methods such as fractional crystallization or column chromatography are not effective. J. Polymer Sci. Polymer Chem. Ed., vol. 22, 341-352 (1984). Tedious column ion exchange or HPLC techniques provide purity of up to 95%, but these techniques are not suitable for large scale processes.
Preparation of a PEG-conjugated product, sometimes referred to as a pegylated product, using impure PEG carboxylic acids results in an mPEG-OH contaminated final product. For lower molecular weight peptides and organic conjugates, removal of the contaminant is very difficult due to the slight difference in molecular weight between the contaminant, mPEG-OH, and the desired polymer conjugate. In addition, using lower purity polymer-carboxylic acid derivatives necessarily reduce the yield of the desired conjugates while adding to manufacturing costs due to the need to undertake tedious and expensive separation steps.
A need exists, therefore, for an improved method of preparing high purity polyalkylene oxide carboxylic acids. The present invention addresses this need.